Base-Stacking-Driven Catalytic Hairpin Assembly: A Nucleic Acid Amplification Reaction Using Electrode Interface as a "Booster" for SARS-CoV-2 Point-of-Care Testing.

Electrochemical DNA (E-DNA) biosensors based on interface-mediated hybridization reactions are promising for point-of-care testing (POCT). However, the low efficiency of target recycle amplification and the steric hindrance at the electrode interface limit their sensing performance. Herein, we propose a base-stacking-driven catalytic hairpin assembly (BDCHA), a nucleic acid amplification reaction strategy, for POCT. The introduction of the base-stacking effect in this strategy increases the thermodynamic stability of the product, thereby effectively improving the recycling efficiency. Also, it enables the interface-mediated hybridization to maintain stability with even fewer bases in the reaction-binding domain, hence minimizing DNA secondary structure formation or intertwining at the electrode surface and ameliorating the steric hindrance limitation. The introduced base-stacking effect makes the electrode serve as a "booster" by integrating the advantages of homogeneous and heterogeneous reactions, giving BDCHA an increased reaction rate of about 20-fold, compared to the conventional catalytic hairpin assembly. As a proof of concept, our BDCHA was applied in constructing a portable E-DNA biosensor for the detection of a SARS-CoV-2 N gene sequence fragment. A simple 30 min one-pot incubation is required, and the results can be readily read on a smartphone, making it portable and user-friendly for POCT.

An Enzymatic Antibiotic Adjuvant Modulates the Infectious Microenvironment to Overcome Antimicrobial Resistance of Pathogens.

The emergence and evolution of antimicrobial resistance (AMR) pose a significant challenge to the current arsenal to fight infection. Antibiotic adjuvants represent an appealing tactic for tackling the AMR of pathogens, however, their practical applications are greatly constrained by the harsh infectious microenvironment. Herein, it is found that silver nanoclusters (Ag NCs) can possess tunable enzymatic activities to modulate infectious microenvironments. Based on this finding, an enzymatic nanoadjuvant (EnzNA) self-assembled from Ag NCs, which is inert under neutral physiological conditions but can readily disassemble into isolated Ag NCs exhibiting biofilm destructive oxidase-mimetic activity in the acidic biofilm microenvironment, is developed. Once internalized into the neutral cytoplasm of bacteria, Ag NCs switch to reveal the thiol oxidase-mimetic activity to suppress ribosomal biogenesis for AMR reversal and evolution inhibition of pathogens. Consequently, EnzNAs revitalize various existing antibiotics against methicillin-resistant Staphylococcus aureus, and potentiate the antibiotic efficacy against biofilm-mediated skin infection and lethal lung infection in mice. These findings highlight the capability of enzyme-mimetic nanomaterials to modulate the infectious microenvironment and potentiate antibiotics, providing a paradigm shift for anti-infection therapy.

Coxsackievirus B: The important agent of hand, foot, and mouth disease.

Hand, foot, and mouth disease (HFMD) is a common pediatric infectious illness caused by enteroviruses (EVs). EV-A serotypes are the main pathogens associated with HFMD. In this study, 213 stool samples from 213 children with severe HFMD in Yunnan, China in 2013, 2015, and 2016 were further analyzed retrospectively for EV-B infection. A total of 70.0% of the specimens tested positive for EV.20 EV serotypes were detected. The predominant serotype was enterovirus A71 (EV-A71, 27.7%), followed by coxsackievirus B4 (CV-B4, 16.4%), CV-A16 (9.9%), CV-B5 (6.6%), and Echovirus 9 (E-9,4.7%). EV-A and EV-B accounted for 45.1% and 41.3%, respectively. Among the positive specimens, 28.6% were CV-Bs. Co-infection was present in 19.3% of these cases. In the study, CV-B5 and the majority of CV-B4 isolates belonged to genotypes VI and C3, respectively. This result indicates that EV-B, especially CV-Bs, might be the important agents associated with HFMD and this knowledge will contribute to the prevention and treatment of the disease.

Gel properties of okara dietary fiber-fortified soy protein isolate gel with/without NaCl.

BACKGROUND: Soy protein isolate (SPI) is widely used as an alternative to animal-based protein, and its gelling property is essential for producing plant protein-based foods. Insoluble dietary fiber has been used to improve the properties of protein gels. RESULTS: Effects of partial replacement of SPI by okara dietary fiber (ODF) on the gelling properties of ODF-fortified SPI gels with and without 0.1 m NaCl were investigated. The presence of ODF hindered the SPI self-aggregation and reduced the surface hydrophobicity of SPI. The presence of ODF reduced the hydrophobic interaction and improved the proportion of disulfide bonds in the gels. In the microstructure, the swollen ODF promoted the local aggregation of SPI at 0.1 m NaCl. Texture profile analysis showed that 5% and 10% ODF improved the SPI gel hardness in the absence of NaCl, whereas only 5% ODF improved the gel hardness at 0.1 m NaCl. The results of low-field nuclear magnetic resonance imaging revealed that ODF shortened the T2 relaxation time of the free water in the gel. The gel of ODF-10 had the highest storage modulus. CONCLUSION: Using an appropriate amount of ODF to replace SPI could improve the quality of SPI gel and increase the dietary fiber content in the product. In addition, the appropriate ratio of ODF/SPI varied in different solution environments. © 2022 Society of Chemical Industry.

Sensitive, Highly Stable, and Anti-Fouling Electrode with Hexanethiol and Poly-A Modification for Exosomal microRNA Detection.

It remains a huge challenge to integrate the sensitivity, stability, reproducibility, and anti-fouling ability of electrochemical biosensors for practical applications. Herein, we propose a self-assembled electrode combining hexanethiol (HT), poly-adenine (poly-A), and cholesteryl-modified DNA to meet this challenge. HT can tightly pack at the electrode interface to form a hydrophobic self-assembled monolayer (SAM), effectively improving the stability and signal-to-noise ratio (SNR) of electrochemical detection. Cholesteryl-modified DNA was immobilized at the electrode through the hydrophobic interaction with HT to avoid the competition between the SAM and the DNA probe on the gold site. Thus, the assembly efficiency and uniformity of the DNA probe as well as the detection reproducibility were increased remarkedly. Poly-A was added on the HT assembled electrode to occupy the unreacted sites of gold to further enhance the anti-fouling ability. The combination of HT and poly-A allows the electrode to ensure favorable anti-fouling ability without sacrificing the detection performance. On this basis, we proposed a dual-signal amplification electrochemical biosensor for the detection of exosomal microRNAs, which showed excellent sensitivity with a detection limit down to 1.46 aM. Importantly, this method has been successfully applied to detect exosomal microRNA-21 in cells and human serum samples, proving its potential utility in cancer diagnosis.

Study on gel properties of lysozyme-free egg white before and after Lactiplantibacillus plantarum fermentation.

BACKGROUND: Isolation of lysozyme from egg white (EW) using ion exchange resin adsorption method generates large quantities of lysozyme-free egg white (LFEW) with poor gelling property. To maximize the applications of LFEW, the effect of Lactiplantibacillus plantarum fermentation on the gel properties of LFEW was investigated in this study. RESULTS: The fermentation efficiency of LFEW with lysozyme removed was significantly improved, and the sugar removal rate (2 g kg-1 Lactiplantibacillus plantarum, 37 °C, 7 h) was more than 90%. Removal of lysozyme resulted in increased stability and surface hydrophobicity of EW. After Lactiplantibacillus plantarum fermentation, the stability of EW decreased, and the average particle size and surface hydrophobicity increased. In addition, by comparing the gel properties of EW and LFEW before and after fermentation at different pH, it was found that the hardness, elasticity, and water holding capacity (WHC) of EW gel increased significantly. The removal of lysozyme effectively improved the WHC and springiness of the EW gel and promoted the formation of a denser network structure with smaller pores. After Lactiplantibacillus plantarum fermentation treatment, LFEW gel hardness decreased, with loose and porous network structure, no browning occurred after autoclaving. CONCLUSION: This study provided the direction and theoretical basis for producing a fermented LFEW gel with pleasing texture and appearance. © 2022 Society of Chemical Industry.

Antibacterial mechanism of linalool emulsion against Pseudomonas aeruginosa and its application to cold fresh beef.

Pseudomonas aeruginosa (P. aeruginosa) is the dominant spoilage bacterium in cold fresh beef. The current strategy is undertaken to overcome the low water solubility of linalool by encapsulating linalool into emulsions. The results of field emission scanning electron microscopy and particle size distribution revealed that the appearance of the bacterial cells was severely disrupted after exposure to linalool emulsion (LE) with an minimum inhibitory concentration (MIC) of 1.5 mL/L. Probes combined with fluorescence spectroscopy were performed to detect cell membrane permeability, while intracellular components (protein and ion leakage) and crystal violet staining were further measured to characterize cell membrane integrity and biofilm formation ability. The results confirmed that LE could destroy the structure of the cell membrane, thereby leading to the leakage of intracellular material and effective removal of biofilms. Molecular docking confirmed that LE can interact with the flagellar cap protein (FliD) and DNA of P. aeruginosa, inhibiting biofilm formation and causing genetic damage. Furthermore, the results of respiratory metabolism and reactive oxygen species (ROS) accumulation revealed that LE could significantly inhibit the metabolic activity of P. aeruginosa and induce oxidative stress. In particular, the inhibition rate of LE on P. aeruginosa was 23.03% and inhibited mainly the tricarboxylic acid cycle (TCA). Finally, LE was applied to preserve cold fresh beef, and the results showed that LE could effectively inhibit the activity of P. aeruginosa and delay the quality change of cold fresh beef during the storage period. These results are of great significance to developing natural preservatives and extending the shelf life of cold fresh beef.

Changes in microbial, physiochemical, and functional properties of pasteurized liquid whole egg during refrigerated storage.

BACKGROUND: Liquid eggs have the advantages of high hygiene security, easy use, and convenient transportation, but their shelf life is only limited to 2~3 weeks. The microbial, physiochemical, and functional properties of pasteurized LWE were investigated in this study to evaluate the quality of pasteurized liquid whole egg (LWE) during refrigerated storage. RESULTS: The tested shelf life of the pasteurized LWE was 16 days when stored at 4 °C. During refrigerated storage, Pseudomonas gradually became the dominant bacterium in LWE following lactic acid bacteria, although the initial number of Pseudomonas after pasteurization was relatively limited (< 10 CFU mL-1 ). A total of 23 strains, including six Pseudomonas strains, were obtained. The pH of pasteurized LWE decreased with the growth of microorganisms, while the content of total volatile basic nitrogen (TVB-N) increased curvilinearly. The average particle size increased almost continuously until the sample reached its shelf life. The functional properties of pasteurized LWE were also reduced after a week of refrigerated storage at 4 °C when the microorganisms in pasteurized LWE entered an exponential growth period and the TVB-N content of pasteurized LWE reached its first peak. CONCLUSION: During refrigerated storage, Pseudomonas was the dominant bacterium in LWE next to lactic acid bacteria. After a week of refrigerated storage at 4 °C, the particle size of LWE increased, while the functional properties of LWE reduced. This study provides a basis for extending the shelf life of liquid egg products in future research. © 2020 Society of Chemical Industry.

The Effect of Ionic Strength on the Formation and Stability of Ovalbumin-Xanthan Gum Complex Emulsions.

Protein-polysaccharide complexes have been widely used to stabilize emulsions, but the effect of NaCl on ovalbumin-xanthan gum (OVA-XG) complex emulsions is unclear. Therefore, OVA-XG complex emulsions with different XG concentrations at pH 5.5 were prepared, and the effects of NaCl on them were explored. The results indicated that the NaCl significantly affected the interaction force between OVA-XG complexes. The NaCl improved the adsorption of proteins at the oil-water interface and significantly enhanced emulsion stability, and the droplet size and zeta potential of the emulsion gradually decreased with increasing NaCl concentrations (0-0.08 M). In particular, 0.08 M NaCl was added to the OVA-0.2% XG emulsion, which had a minimum droplet size of 18.3 µm. Additionally, XG as a stabilizer could improve the stability of the emulsions, and the OVA-0.3% XG emulsion also exhibited good stability, even without NaCl. This study further revealed the effects of NaCl on emulsions, which has positive implications for the application of egg white proteins in food processing.

Nondestructive identification and classification of starch types based on multispectral techniques coupled with chemometrics.

Starch is the main source of energy and nutrition. Therefore, some merchants often illegally add cheaper starches to other types of starches or package cheaper starches as higher priced starches to raise the price. In this study, 159 samples of commercially available wheat starch, potato starch, corn starch and sweet potato starch were selected for the identification and classification based on multispectral techniques, including near-infrared (NIR), mid-infrared (MIR) and Raman spectroscopy combined with chemometrics, including pretreatment methods, characteristic wavelength selection methods and classification algorithms. The results indicate that all three spectral techniques can be used to discriminate starch types. The Raman spectroscopy demonstrated superior performance compared to that of NIR and MIR spectroscopy. The accuracy of the models after characteristic wavelength selection is generally superior to that of the full spectrum, and two-dimensional correlation spectroscopy (2D-COS) achieves better model performance than other wavelength selection methods. Among the four classification methods, convolutional neural network (CNN) exhibited the best prediction performance, achieving accuracies of 99.74 %, 97.57 % and 98.65 % in NIR, MIR and Raman spectra, respectively, without pretreatment or characteristic wavelength selection.

Effects of different temperatures on the physicochemical characteristics, microstructure and protein structure of preserved egg yolk.

To clarify the mechanism of lower temperatures promoted the solidification of preserved egg yolk, the effects of temperature (4 °C, 10 °C and 25 °C) on the physicochemical properties, microstructure and protein structure of preserved egg yolk were studied. Results showed that the exterior egg yolk (EEY) exhibited higher pH, hardness and free sulfhydryl content at low-temperature pickling. The microstructure showed that the EEY gradually formed a denser gel network structure at lower temperatures. Electrophoresis results and Fourier transform infrared spectroscopy (FTIR) indicated that there were different degrees of protein degradation and cross-linking of proteins in the IEY (the interior egg yolk) and EEY and the decrease of ß-sheets in the secondary structure was accompanied by an increase of ß-turns during the formation of egg yolk gels. These results indicated that egg yolk solidification was faster and denser gel structure at 4 °C and 10 °C.

Recent trends in design of healthier fat replacers: Type, replacement mechanism, sensory evaluation method and consumer acceptance.

In recent years, with the increasing awareness of consumers about the relationship between excessive fat intake and chronic diseases, such as obesity, heart disease, diabetes, etc., the demand for low-fat foods has increased year by year. However, a simple reduction of fat content in food will cause changes in physical and chemical properties, physiological properties, and sensory properties of food. Therefore, developing high-quality fat replacers to replace natural fats has become an emerging trend, and it is still a technical challenge to completely simulate the special function of natural fat in low-fat foods. This review aims to provide an overview of development trends of fat replacers, and the different types of fat replacers, the potential fat replacement mechanisms, sensory evaluation methods, and their consumer acceptance are discussed and compared, which may provide a theoretical guidance to produce fat replacers and develop more healthy low-fat products favored by consumers.

Characteristics of hen egg white lysozyme, strategies to break through antibacterial limitation, and its application in food preservation: A review.

Hen egg white lysozyme (HEWL) is used as a food additive in China due to its outstanding antibacterial properties. It is listed as GRAS grade (generally recognized as safe) by the United States Food and Drug Administration (FDA, US) and has been extensively researched and used in food preservation. And the industrial production of HEWL already been realized. Given the complex food system that can affect the antibacterial activity of HEWL, and the limitations of HEWL itself on Gram-negative bacteria. Based on the structure and main biological characteristics of HEWL, this paper focuses on reviewing methods to enhance the stability and antibacterial properties of HEWL. Immobilization tactics such as chemically driven self-assembly, embedding and adsorption address the restriction of poor HEWL antibacterial activity effected by external factors. Both intermolecular and intramolecular modification strategies break the bactericidal deficiencies of HEWL itself. It also comprehensively analyzes the current application status and future prospects of HEWL in the food preservation. There was limited research on the biological methods in modifying HEWL. If the HEWL is genetically engineered, it can broaden its antimicrobial spectrum, improve its other biological activities, so as to further expand its application in the food industry. At present, research on HEWL mainly focused on its antibacterial properties, whereas its application in anti-inflammatory and antioxidant effects also presented great potential.

Comparative key aroma compounds and sensory correlations of aromatic coconut water varieties: Insights from GC × GC-O-TOF-MS, E-nose, and sensory analysis.

Aroma is a key criterion in evaluating aromatic coconut water. A comparison regarding key aroma compounds and sensory correlations was made between Thailand Aromatic Green Dwarf (THD) and Cocos nucifera L. cv. Wenye No. 4 coconut water using E-nose and GC × GC-O-TOF-MS combined with chemometrics. Twenty-one volatile components of coconut water were identified by GC × GC-O-TOF-MS, and 5 key aroma compounds were analyzed by relative odor activity value and aroma extract dilution analysis. Moreover, the combination of the E-nose with orthogonal partial least squares was highly effective in discriminating between the two coconut water samples and screened the key sensors responsible for this differentiation. Additionally, the correlation between volatile compounds and sensory properties was established using partial least squares. The key aroma compounds of coconut water exhibited positive correlations with the corresponding sensory properties.

Mechanism of ultrasonic enhancement of the gelling properties of salted ovalbumin-cooked soybean isolate hybrid gels.

The influence of ultrasonic processing on the physicochemical characteristics, microstructure, and intermolecular forces of the hybrid gels obtained by heating the mixtures of different ratios of salted ovalbumin (SOVA)-cooked soybean protein isolate (CSPI) was investigated. With the growth of SOVA addition, ζ-potential in absolute value, cohesiveness, water-holding capacity (WHC), surface hydrophobicity, and the content of soluble protein of the hybrid gels decreased (P < 0.05), while the hardness, T2 relaxation time of the hybrid gels increased (P < 0.05). And the compactness of the network structure of the hybrid gel increased with the increase of SOVA addition. After being treated with ultrasound, significant increases (P < 0.05) of ζ-potential in absolute value, cohesiveness, WHC, and surface hydrophobicity of the hybrid gels were observed. In general, ultrasonic processing is one of the effective means to improve the gel properties of SOVA-CSPI hybrid gels.

Influenza a Neuraminidase-Based Bivalent mRNA Vaccine Induces Th1-Type Immune Response and Provides Protective Effects in Mice.

Vaccines are one of the most effective means of preventing influenza A, typically containing the hemagglutinin (HA) of the influenza A virus. However, antigenic drift and shift of the influenza A virus can lead to instability in vaccine efficacy. Compared to HA, the antigenic variation rate of neuraminidase (NA) is slower. In traditional inactivated influenza vaccines, although they contain a certain amount of NA, there are significant differences between different batches, which cannot consistently induce NA-based immune responses. Therefore, NA is often overlooked in vaccine development. In this study, we report an mRNA vaccine encoding the NA of two strains of influenza A virus. The experimental results demonstrated that when matched with the viral strain, this mRNA vaccine induced high levels of neutralizing antibodies, providing a protective effect to mice in viral challenge experiments, and this immune response was shown to be biased towards the Th1 type. In summary, this study demonstrates that NA is a promising potential antigen, providing new insights for the development of influenza A virus vaccines.

Integrated Analysis of Metabolomics Combined with Network Pharmacology and Molecular Docking Reveals the Effects of Processing on Metabolites of Dendrobium officinale.

Dendrobium officinale (D. officinale) is a precious medicinal species of Dendrobium Orchidaceae, and the product obtained by hot processing is called "Fengdou". At present, the research on the processing quality of D. officinale mainly focuses on the chemical composition indicators such as polysaccharides and flavonoids content. However, the changes in metabolites during D. officinale processing are still unclear. In this study, the process was divided into two stages and three important conditions including fresh stems, semiproducts and "Fengdou" products. To investigate the effect of processing on metabolites of D. officinale in different processing stages, an approach of combining metabolomics with network pharmacology and molecular docking was employed. Through UPLC-MS/MS analysis, a total of 628 metabolites were detected, and 109 of them were identified as differential metabolites (VIP ≥ 1, |log2 (FC)| ≥ 1). Next, the differential metabolites were analyzed using the network pharmacology method, resulting in the selection of 29 differential metabolites as they have a potential pharmacological activity. Combining seven diseases, 14 key metabolites and nine important targets were screened by constructing a metabolite-target-disease network. The results showed that seven metabolites with potential anticoagulant, hypoglycemic and tumor-inhibiting activities increased in relative abundance in the "Fengdou" product. Molecular docking results indicated that seven metabolites may act on five important targets. In general, processing can increase the content of some active metabolites of D. officinale and improve its medicinal quality to a certain extent.

Cell-Free Supernatant of Lactiplantibacillus plantarum 90: A Clean Label Strategy to Improve the Shelf Life of Ground Beef Gel and Its Bacteriostatic Mechanism.

Lactic acid bacteria metabolites can be used as a clean-label strategy for meat products due to their "natural" and antibacterial properties. In this study, the feasibility of using cell-free supernatant of Lactiplantibacillus plantarum 90 (LCFS) as a natural antibacterial agent in ground beef was investigated. The sensitivity of LCFS to pH, heat and protease, as well as the changes of enzyme activities of alkaline phosphatase (AKP) and Na+/K+-ATP together with the morphology of indicator bacteria after LCFS treatment, were analyzed to further explore the antibacterial mechanism of LCFS. The results showed that the addition of 0.5% LCFS inhibited the growth of microorganisms in the ground beef gel and extended its shelf-life without affecting the pH, cooking loss, color and texture characteristics of the product. In addition, the antibacterial effect of LCFS was the result of the interaction of organic acids and protein antibacterial substances in destroying cell structures (cell membrane, etc.) to achieve the purpose of bacteriostasis. This study provides a theoretical basis for the application of LCFS in meat products and a new clean-label strategy for the food industry.

Dual amplified electrochemical sensing coupling of ternary hybridization-based exosomal microRNA recognition and perchlorate-assisted electrocatalytic cycle.

Exosomal microRNA (miRNA) are important biomarkers for liquid biopsy, and display clinical molecular signatures for cancer diagnosis. Although advanced detection methods have been established to detect exosomal miRNAs, they are faced with certain challenges. Therefore, we aimed to establish a dual amplification-based electrochemical method for detecting exosomal miRNA. This method combined a two-hairpins-based ternary hybridization structure (thTHS)-initiated single-stranded DNA (ssDNA) amplification reaction (ssDAR) and sodium perchlorate (NaClO4)-assisted electrocatalytic cycle. Two DNA hairpins were designed to hybridize with target miRNA, forming thTHS. Next, ssDAR was triggered by thTHS to produce long ssDNA on magnetic beads. The long ssDNA, complementary to the signal probes, was subsequently released onto a methylene blue (MB)-labeled double-stranded DNA-modified electrode for strand displacement reaction. This led to a quantitative change in MB and a change in electrocatalytic reduction current from the electrocatalytic cycle of MB-ferricyanide. An amplified electrocatalytic reduction current was produced by adding NaClO4 to the electrocatalytic system, which substantially improved the signal response range and detection sensitivity. Ultimately, exosomal miRNA detection was achieved by recording changes in the electrocatalytic reduction current before and after miRNA addition. This electrochemical method exhibited a sensitive concentration response with a detection limit of 45 aM and selective miRNA recognition, and successfully used to detect exosomal miRNA derived from cells and serum. Additionally, this method exhibited better discrimination ability between patients with breast cancer (BC) and those people without BC (patients with benign breast disease and healthy people), providing a promising strategy for detecting and monitoring cancer biomarkers.

Screening of a new candidate coxsackievirus B1 vaccine strain based on its biological characteristics.

Coxsackievirus B1 (CVB1) is one of the significant pathogens causing viral myocarditis, hand, foot, and mouth disease (HFMD), and aseptic meningitis, and it has been associated with type 1 diabetes (T1DM). No effective antiviral drugs against CVB1 infection or preventive vaccines are available. Due to the success of two inactivated vaccines against enterovirus 71 and poliovirus, an inactivated Vero cell-based CVB1 vaccine could be developed. In this study, we isolated a high-growth CVB1 virus strain KM7 in Vero cells and developed a Vero-adapted vaccine candidate strain KM7-X29 via three rounds of plaque purification and serial passages. The KM7-X29 strain was grouped into the GII sub-genotype, which belonged to the Chinese epidemic strain and grew to a titer of more than 107 CCID50/ml in Vero cells. The inactivated CVB1 vaccine produced by the KM7-X29 strain induced an effective neutralizing antibody response in BALB/c mice, and maternal antibodies were able to provide a 100% protective effect against lethal challenges with a CVB1 strain in suckling BALB/c mice. Thus, the KM7-X29 strain might be used as a new candidate coxsackievirus B1 vaccine strain. The neonatal murine model of CVB1 infection will contribute to the development of the CVB1 vaccine.